This invention relates to a nucleic acid molecule which encodes human NIM1 kinase and to the use of the nucleic acid molecule and the protein it encodes in the characterization, diagnosis, prevention, and treatment of brain disorders and cancers, particularly breast cancer.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the protein kinases of nematode, fly, rat, and man have common chemical and structural features and modulate the same general cellular activity. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Protein kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein phosphorylation is the main strategy for controling activities of eukaryotic cells. It is estimated that more than 1000 of the 10,000 proteins active in a typical mammalian cell are phosphorylated. The high energy phosphate which drives activation is generally transferred from adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation is roughly analogous to turning on a molecular switch, and it occurs in response to extracellular signals (mediated by such molecules as hormones, neurotransmitters, growth and differentiation factors), cell cycle checkpoints, and environmental or nutritional stresses. When the switch goes on, the appropriate protein kinase activates a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.
The protein kinases comprise the largest known protein group, a superfamily of enzymes with widely varied functions and specificities. They are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With regard to substrates, the protein kinases may be roughly divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity and phosphorylate both threonine and tyrosine residues.
Protein kinases may be categorized into families by the different amino acid residues (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the catalytic domain. These residues allow the regulation of each kinase as it recognizes and interacts with its target protein. Almost all kinases contain a similar 250-300 amino acid catalytic domain with 11 subdomains distributed across two lobes. The N-terminal lobe, which contains subdomains I-IV, binds and orients the ATP donor molecule. The larger C terminal lobe, which contains subdomains VIA-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the N and C terminal lobes.
Each of the 11 subdomains contain specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved (Hardie and Hanks (1995) The Protein Kinase Facts Books, Vol I, Academic Press, San Diego Calif., pp. 7-20). In particular, two protein kinase signature sequences have been identified in the kinase domain, the first containing an active site lysine residue involved in ATP binding, and the second containing an aspartate residue important for catalytic activity. If a protein is found to contain the two protein kinase signatures, the probability of that protein being a protein kinase is close to 100% (MOTIFS search program, Genetics Computer Group, Madison Wis.; Bairoch et al. (1996) Nucleic Acids Res 24:189-196).
STK Families
The second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic ADP ribose, arachidonic acid, diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) are important members of the STK family. cAMP is an intracellular mediator of hormone action in all prokaryotic and animal cells that have been studied. Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone resorption, and regulation of heart rate and force of heart muscle contraction. PKA is found in all animal cells and is thought to account for the effects of cAMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher et al. (1994) Harrison""s Principles of Internal Medicine, McGraw-Hill, New York N.Y., pp. 416-431 and 1887).
Calcium-calmodulin (CaM) dependent protein kinases are also members of STK family. Calmodulin is a calcium receptor that mediates many calcium regulated processes by binding to target proteins in response to the binding of calcium. The principle target protein in these processes is CaM dependent protein kinases (CaMK). CaMK are involved in regulation of smooth muscle contraction, glycogen breakdown (phosphorylase kinase), and neurotransmission (CaMK I and CaMK II). CaMK I phosphorylates a variety of substrates including the neurotransmitter related proteins synapsin I and II, the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR (Haribabu et al. (1995) EMBO J 14:3679-86). CaMK II also phosphorylates synapsin at different sites and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase. Many of the CAMK are activated by phosphorylation in addition to binding to CaM. CaMK may autophosphorylate or be phosphorylated by another kinase as part of a xe2x80x9ckinase cascadexe2x80x9d.
Another ligand-activated protein kinase is 5xe2x80x2-AMP-activated protein kinase (AMPK; Dyck et al. (1996) J Biol Chem 271:17998-17803). Mammalian AMPK is a regulator of fatty acid and sterol synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways to cellular stresses such as heat shock and depletion of glucose and ATP. AMPK is a heterotrimeric complex comprised of a catalytic alpha subunit and two non-catalytic beta and gamma subunits that are believed to regulate the activity of the alpha subunit. Subunits of AMPK have a much wider distribution in non-lipogenic tissues such as brain, heart, spleen, and lung than expected: This distribution suggests that its role may extend beyond regulation of lipid metabolism alone.
The mitogen-activated protein kinases (MAPK) are also members of the STK family, and they regulate intracellular signaling pathways. MAPK mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli (Egan and Weinberg (1993) Nature 365:781-783). MAP kinase signaling pathways are present in mammalian cells as well as in yeast. The extracellular stimuli which activate mammalian pathways include epidermal growth factor, ultraviolet light, hyperosmolar medium, heat shock, endotoxic lipopolysaccharide, and pro-inflammatory cytokines such as tumor necrosis factor and interleukin-1. Altered MAPK expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.
Proliferation-related kinase (PRK) is a serum/cytokine inducible STK that is involved in regulation of the cell cycle and cell proliferation in human megakaroytic cells (Li et al. (1996) J Biol Chem 271:19402-8). PRK is related to the polo family of STKs implicated in cell division. PRK is downregulated in lung tumor tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation.
The cyclin-dependent protein kinases (CDKs) are another group of STKs that control the progression of cells through the cell cycle. Cyclins are small regulatory proteins that act by binding to and activating CDKs which then trigger various phases of the cell cycle by phosphorylating and activating selected proteins involved in the mitotic process. CDKs are unique in that they require multiple inputs to become activated. In addition to the binding of cyclin, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue.
The discovery of nucleic acid molecules encoding a human NIM1 kinase (hNIM) provides new compositions which are useful in the characterization, diagnosis, prevention, and treatment of brain disorders and cancers, particularly breast cancer.
The invention is based on the discovery of a substantially purified nucleic acid molecule encoding a human NIM1 kinase which satisfies a need in the art by providing compositions useful in the characterization, diagnosis, prevention, and treatment of brain disorders and cancers, particularly breast cancer.
The invention provides a substantially purified nucleic acid molecule which encodes the human NIM1 kinase comprising SEQ ID NO:2. The invention also provides a composition comprising SEQ ID NO:1 or a fragment or a complement thereof. The invention further provides a mammalian variant of the nucleic acid molecule selected from SEQ ID NOs:24-30. The invention still further provides a fragment of at least 18 consecutive nucleotides selected from about nucleotide 414 to about 1414 of SEQ ID NO:1, SEQ ID NOs:3-30, or the complements thereof. In one aspect, the invention provides a substrate containing at least one of these fragments. In a second aspect, the invention provides a probe comprising the fragment which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention provides a method for detecting a nucleic acid molecule in a sample, the method comprising the steps of hybridizing a probe or complementary nucleic acid molecule to at least one nucleic acid in a sample, forming a hybridization complex; and detecting the hybridization complex, wherein the presence of the hybridization complex indicates the presence of the nucleic acid molecule in the sample. In one aspect, the method further comprises amplifying the nucleic acids of the sample prior to hybridization. The nucleic acid molecule or a fragment or a complement thereof may comprise an element on an array.
The invention also provides a method for using a nucleic acid molecule or a fragment or a complement thereof to screen a library of molecules or compounds to identify at least one ligand which specifically binds the nucleic acid molecule, the method comprising combining the nucleic acid molecule with a library of molecules or compounds under conditions allowing specific binding, and detecting specific binding to the nucleic acid molecule, thereby identifying a ligand which specifically binds the nucleic acid molecule. In one aspect, the libraries of molecules and compounds include DNA and RNA molecules, peptides, PNAs, proteins, and the like.
The invention further provides an expression vector containing at least a fragment of the nucleic acid molecule which is contained within a host cell. The invention still further provides a method for producing a protein, the method comprising the steps of culturing the host cell under conditions for the expression of the protein and recovering the protein from the host cell culture.
The invention provides an isolated and purified protein comprising SEQ ID NO:2 or a portion thereof. Additionally, the invention provides a pharmaceutical composition comprising a substantially purified protein or a portion thereof in conjunction with a pharmaceutical carrier.
The invention provides a method for using at least a portion of the protein to produce antibodies. The invention also provides a method for using a protein to screen a library of molecules or compounds to identify at least one ligand which specifically binds the protein, the method comprising combining the protein with the library of molecules or compounds under conditions allowing specific binding, and detecting bound protein, thereby identifying a ligand which specifically binds the protein. Such libraries of molecules and compounds include agonists, antagonists, antibodies, DNA and RNA molecules, immunoglobulins, drug compounds, mimetics, peptides, pharmaceutical agents, and other ligands. The invention further provides an analogous method which uses the protein to purify a ligand. The method involves combining the protein with a sample under conditions to allow specific binding, recovering the bound protein, and separating the protein from the ligand to obtain purified ligand.
The invention additionally provides antibodies identified by screening methods using or antibodies produced against the human NIM1 kinase. A method of preparing an antibody comprising immunizing an animal with the human NIM1 kinase or an antigenically-effective portion thereof under conditions to elicit an antibody response; isolating animal antibodies; and screening the isolated antibodies with NIM1 kinase thereby identifying an antibody specifically binds Nim1 kinase. In one aspect these antibodies are useful as diagnostic compositions in identification of brain disorders and cancers. In another aspect, the antibody may be administered as a pharmaceutical composition to treat brain disorders and cancers associated with the overexpression of human NIM1 kinase.
The further provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the natural nucleic acid. The invention also provides a method for using a nucleic acid molecule to produce a mammalian model system, the method comprising constructing a vector containing the nucleic acid molecule selected from SEQ ID NOs:1 and 3-30; transforming the vector into an embryonic stem cell; selecting a transformed embryonic stem; microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst; transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the nucleic acid molecule in its germ line; and breeding the chimeric mammal to produce a homozygous, mammalian model system.